含当归芍药散简方脑脊液对神经细胞的保护作用

目的:探讨含当归芍药散简方脑脊液的神经保护作用及其可能的机制.方法:采用FeSO4和H202作用产生自由基的方法诱导建立PC12细胞氧化损伤模型,分别用当归芍药散简方(0.135 g·mL-1)ig家兔(1.5 g·kg-1),于1,14 d时取脑脊液添加于培养液中(每组分5％,10％,20％3个体积分数)处理PC12细胞,MTT法铡定细胞活性,硫代巴比妥酸(TBA)比色法测定丙二醛(MDA)含量,黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活力,甲基百里香酚蓝(MTB)比色法测定细胞内Ca1+水平,免疫细胞化学法测定α7烟碱型乙酰胆碱受体(nAchR)阳性表达,BCA蛋白定量及斑点印迹法测定α7nAchR亚单位水平.结果:含20％当归芍药散简方脑脊液对PC12细胞活性(以吸光度表示,A)1 d组(1.241±0.117)和14d组(1.297±0.213)与损伤组(0.986±0.051)比较能明显增加PC12细胞活性(P＜0.05,P＜0.01)、显著提高SOD活力(19.48±0.34),(19.52±0.33)U·mL-1对(18.18±0.12)U·mL-1(P＜0.01),有效降低MDA含量及细胞内Ca2+水平均降低,与损伤组比较P＜0.01；明显上调α7nAChR的表达,14 d组与1d组比较有显著性差异(P＜0.05).结论:当归芍药散简方对FeSO4和H2O2诱导建立PC12细胞氧化损伤模型具有保护作用,其机制与保护α7nAchR有关.

The Protective Effect of Cerebrospinal Fluid Containing Optimized Danggui Shaoyao San on PC12 Cell Injury Induced by H2O2/Fe2+

Objective: To explore the protective effect of cerebrospinal fluid(CSF) with the optimized Danggui Shaoyao San(FBD) and its underlying mechanisms.Method: PC12 cells were treated with H2O2 / Fe2+ to reproduce the cell injury model.The rabbits were randomly divided into 1 d group and 14 d group and they were given FBD by intragastric administrate.PC12 cells were treated with different concentrations of CSF containing FBD(5%,10%,20%).Cell survival was assessed by Thiazzolyl blue(MTT) assay.The content of maleic diadehyde(MDA) and superoxide dismutase(SOD) activity were determined respectively by thiobarbituric acid and xanthine oxidation enzyme.The Ca2+content within cell was measured by MTB colorimetry.The protein expression of α7 nicotinic acetylcholine receptor(nAchR) was examined by using immunocytochemical detecting kit,bicinchoninic acid(BCA) protein kit and Dot blot assay.Result: The 20% group for 1 day(1.241±0.117) and 14 days(1.297±0.213) of FBD improved the survival of the PC12.The content of SOD increased from(18.18±0.12) U·mL-1 in the 20% group for 1 d to(19.48±0.34)U·mL-1 and 14 d(19.52±0.33) U·mL-1(P<0.01).In addition,α7nAChR in PC12 cells was significantly up-regulated.The expression of α7nAChR in 20% group for 14 d(0.161±0.024) was significantly higher than that in the group for 1 d(0.132±0.021)(P<0.05).These groups reduced MDA and Ca2+within cell from(3.26±0.13)mmol·L-1,(74.89±3.66)nmol/105 cell and(2.77±0.05)mmol·L-1,(74.25±2.64)nmol/105 cell to(4.41±0.23)mmol·L-1,(82.42±4.25) nmol/105 cells).Conclusion: The CSF containing FBD can prevent from the neurotoxicity induced by H2O2 / Fe2+.It may be related to α7nAchR up-regulation.